Detection of a molecular marker formed in the blood in response to activation of the coagulation-fibrinolytic system is an important factor for the early diagnosis of disseminated intravascular coagulation (DIC) syndrome, as well as checking of condition thereof. Particularly, soluble fibrin monomer complex (SFMC) has been clinically employed as a marker for detecting initial hypercoagulability.
Thrombin, which has been formed through activation in a blood vessel, cuts the N-terminal of an Aα-chain of fibrinogen to thereby form a desAA fibrin monomer, and further cuts the N-terminal of a Bβ-chain thereof to thereby form a desAABB fibrin monomer. The thus-formed fibrin monomer forms a complex with fibrinogen or others in the blood, and the complex (i.e., SFMCs) circulates in the blood. As has been known, thrombogenesis can be detected in an early stage through detecting the SFMC.
Hitherto, a variety of specific antibodies and immunological assay methods for detecting SFMC have been reported. For example, G. Soe et al. have reported an IF-43 antibody as a monoclonal antibody which recognizes a conformation-change occurring in the E domain during formation of a fibrin-fibrinogen complex from a fibrin monomer and fibrinogen. The epitope recognized by the IF-43 antibody is present at an amino acid sequence of the 17th to 78th amino acid residues in the N-terminal region of the Aα chain (SEQ ID NO: 1). The IF-43 antibody is characterized in that it does not act on fibrinogen, fibrin monomer, or fibrinogen-degradation products by plasmin or fibrin-degradation products by plasmin, and thus reflects the blood coagulation system; WO95/012617 and Soe, et al., Blood 88: 2109-2117 (1996).
However, a soluble fibrin assay reagent employing the IF-43 antibody (latro SF, Iatron) is known to act on a complex formed through reaction between fibrin monomer-degradation products by plasmin (fibrin fragment X, Y, or E) and fibrinogen, as well as on complex formed through reaction between fibrin monomer-degradation products by plasmin and fibrinogen-degradation products by plasmin (fragment X, Y, or D); JP (kokai) 2004-53359. Thus, since the IF-43 antibody also acts on the plasmin-digested soluble fibrin, the antibody cannot be considered as an antibody reflecting exclusively the coagulation system.
Also, an antibody which recognizes an amino acid sequence in the N-terminal region of the fibrinogen Aα chain (SEQ ID NO: 1) which is formed by cutting the Aα chain with thrombin has been reported. Specifically, Scheefers-Borchell et al. previously produced an antibody specific to soluble fibrin through immunization with a synthetic hexapeptide GPRVVE (SEQ ID NO: 2), which is identical to the amino acid sequence of the N-terminal region of fibrin, Scheefers-Borchell, et al., PNAS 82: 7091-7095 (1985). A. Hamano et al. previously produced an antibody (F405) specific to soluble fibrin using a fibrin monomer prepared by treating fibrinogen with batroxobin, as an immunogen; WO98/59047 and Hamano, et al., Clin. Chim. Acta 318: 25-32 (2002).
However, since the epitope recognized by these antibodies is an N-terminal amino acid sequence site which is formed through an action of thrombin on the Aα chain, these antibodies are considered to act on fibrin monomer-degradation products by plasmin, complexes thereof, and XDP fractions (DY, DXD, etc., cross-linked fibrin-degradation products by plasmin). Therefore, these antibodies are considered not to exclusively reflect the coagulation system, but to reflect both the coagulation and the fibrinolytic systems.
Furthermore, a monoclonal antibody which reacts with a peptide formed of the amino acid sequence of the 148th to 161st amino acid residues of a fibrinogen Aα chain has been reported; Japan (kokai) 2-028197. Since the recognition site of the antibody is not in the C-terminal side of the Aα chain (SEQ ID NO: 1) which is digested with plasmin, the antibody is considered to react with plasmin-digested soluble fibrin or cross-linked fibrin-degradation products by plasmin. Thus, the antibody cannot be considered as an antibody reflecting exclusively the coagulation system.
As described above, in many cases, conventional antibodies against a soluble fibrin formed through hypercoagulability also recognize a plasmin-digested soluble fibrin or cross-linked fibrin-degradation products by plasmim formed through the fibrinolytic system. Hitherto, no antibody has been known that specifically recognizes soluble fibrin on which plasmin has not acted, and no method has been reported that reflects exclusively the coagulation system.    [Patent Document 1] International Patent Publication No. 95/012617 Pamphlet    [Patent Document 2] Japanese Patent Application Laid-Open (kokai) No. 2004-53359    [Patent Document 3] International Patent Publication No. 98/59047 Pamphlet    [Patent Document 4] Japanese Patent Application Laid-Open (kokai) No. 2-028197    [Non-Patent Document 1] G. Soe., et al, Blood. 88, 2109-2117, 1996.    [Non-Patent Document 2] U. SCHEEFERS-BORCHEL et al, Proc. Natl. Acad. Sci. USA. 82, 7091-7095, 1985.    [Non-Patent Document 3] A. Hamano et al, Clinica Chimica Acta. 318, 25-32, 2002.